Tissue repair fabric

ABSTRACT

A prosthesis, which, when implanted into a mammalian patient, serves as a functioning replacement for a body part, or tissue structure, and will undergo controlled biodegradation occurring concomitantly with bioremodeling by the patients living cells. The prosthesis is treated so that it is rendered non-antigenic so as not to elicit a significant humoral immune response. The prosthesis, in its various embodiments, thus has dual properties. First, it functions as a substitute body part, and second, it functions as bioremodeling template for the ingrowth of host cells.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the field of implantable biological prostheses. Thepresent invention is a non-antigenic, resilient, completelybioremodelable, biocompatible tissue prosthesis which can be engineeredinto a variety of shapes and used to repair, augment, or replacemammalian tissues and organs. Each layer of the prosthesis is graduallydegraded and remodeled by the host's cells which replace the implantedprosthesis in its entirety to restore structure and function and isuseful for organ repair and reconstruction. Thus, the prosthesis acts asa template by which the host's cells will remodel themselves through aprocess that will replace the prosthetic collagen molecules with theappropriate host cells in order to restore and replace the original hosttissue or organ.

2. Brief Description of the Background of the Invention

Despite the growing sophistication of medical technology, repairing andreplacing damaged tissues remains a frequent, costly, and seriousproblem in health care. Currently implantable prostheses are made from anumber of synthetic and treated natural materials. The ideal prostheticmaterial should be chemically inert, non-carcinogenic, capable ofresisting mechanical stress, capable of being fabricated in the formrequired, and sterilizable, yet not be physically modified by tissuefluids, excite an inflammatory or foreign body reaction, induce a stateof allergy or hypersensitivity, or, in some cases, promote visceraladhesions (Jenkins S. D., et al. Surgery 94(2):392–398, 1983).

For example, body wall defects that cannot be closed with autogenoustissue due to trauma, necrosis or other causes require repair,augmentation, or replacement with synthetic mesh. In reinforcing orrepairing abdominal wall defects, several prosthetic materials have beenused, including tantalum gauze, stainless steel mesh, DACRON®, ORLON®,FORTISAN®, nylon, knitted polypropylene (MARLEX®), microporousexpanded-polytetrafluoroethylene (GORE-TEX®), dacron reinforced siliconerubber (SILASTIC®), polyglactin 910 (VICRYL®), polyester (MERSWENE®),polyglycolic acid (DEXON®), processed sheep dermal collagen (PSDC®),crosslinked bovine pericardium (PERI-GUARD®), and preserved human dura(LYODURA®). No single prosthetic material has gained universalacceptance.

The major advantages of metallic meshes are that they are inert,resistant to infection and can stimulate fibroplasia. Their majordisadvantage is the fragmentation that occurs after the first year ofimplantation as well as the lack of malleability. Synthetic meshes havethe advantage of being easily molded and, except for nylon, retain theirtensile strength in the body. European Patent No. 91122196.8 to Krajicekdetails a triple-layer vascular prosthesis which utilizesnon-resorbable, synthetic mesh as the center layer. The synthetictextile mesh layer is used as a central frame to which layers ofcollagenous fibers can be added, resulting in the tri-layered prostheticdevice. The major disadvantage of a non-resorbable synthetic mesh islack of inertness, susceptibility to infection, and interference withwound healing.

In contrast to the non-resorbable mesh disclosed in Krajicek (E.P. No.91122196.8), absorbable synthetic meshes have the advantage ofimpermanence at the site of implantation, but often have thedisadvantage of losing their mechanical strength, because of dissolutionby the host, prior to adequate cell and tissue ingrowth.

The most widely used material for abdominal wall replacement and forreinforcement during hernia repairs is MARLEX®; however, severalinvestigators reported that with scar contracture, polypropylene meshgrafts became distorted and separated from surrounding normal tissue ina whorl of fibrous tissue. Others have reported moderate to severeadhesions when using MARLEX®.

GORE-TEX® is currently believed to be the most chemically inert polymerand has been found to cause minimal foreign body reaction whenimplanted. A major problem exists with the use ofpolytetrafluoroethylene in a contaminated wound as it does not allow forany macromolecular drainage, which limits treatment of infections.

Collagen first gained utility as a material for medical use because itwas a natural biological prosthetic substitute that was in abundantsupply from various animal sources. The design objectives for theoriginal collagen prosthetics were the same as for synthetic polymerprostheses; the prosthesis should persist and essentially act as aninert-material. With these objectives in mind, purification andcrosslinking methods were developed to enhance mechanical strength anddecrease the degradation rate of the collagen (Chvapil, M., et al (1977)J. Biomed. Mater. Res. 11: 297–314; Kligman, A. M., et al (1986) J.Dermatol. Surg. Oncol. 12 (4): 351–357; Roe, S. C., et al. (1990).Artif. Organs. 14: 443–448. Woodroff, E. A. (1978). J. Bioeng. 2: 1–10).Crosslinking agents originally used include glutaraldehyde,formaldehyde, polyepoxides, diisocyanates (Borick P. M., et al. (1964)J. Pharm. Sci. 52: 1273–1275), and acyl azides. Processed dermal sheepcollagen has been studied as an implant for a variety of applications.Before implantation, the sheep dermal collagen is typically tanned withhexamethylenediisocyanate (van Wachem, P. B., et al. Biomaterials12(March):215–223, 1991) or glutaraldehyde (Rudolphy, V. J., et al. AnnThorac Surg 52:821–825, 1991). Glutaraldehyde, probably the most widelyused and studied crosslinking agent, was also used as a sterilizingagent. In general, these crosslinking agents generated collagenousmaterial which resembled a synthetic material more than a naturalbiological tissue, both mechanically and biologically.

Crosslinking native collagen reduces the antigenicity of the material(Chvapil, M. (1980) Reconstituted collagen. pp. 313–324. In: Viidik, A.,Vuust, J. (eds), Biology of Collagen. Academic Press, London; Harjula,A., et al. (1980) Ann. Chir. Gynaecol. 69: 256–262.) by linking theantigenic epitopes rendering them either inaccessible to phagocytosis orunrecognizable by the immune system. There are many known methods ofcrosslinking collagenous materials. U.S. Pat. No. 5,571,216 detailsseveral methods of achieving crosslinking through the heating andjoining of free ends of collagen tendrils. U.S. Pat. No. 5,263,983 toYoshizato details crosslinking by treating collagenous composites with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.Glutaraldehyde is also employed as a reagent in crosslinking (See U.S.Pat. No. 4,787,900 to Yannas; U.S. Pat. No. 4,597,762 to Walter).However, data from studies using glutaraldehyde as the crosslinkingagent are hard to interpret since glutaraldehyde treatment is also knownto leave behind cytotoxic residues (Chvapil, M. (1980), supra; Cooke,A., et al. (1983) Br. J. Exp. Path. 64: 172–176; Speer, D. P., et al.(1980) J. Biomed. Mater. Res. 14: 753–764; Wiebe, D., et al. (1988)Surgery. 104: 26–33). It is, therefore, possible that the reducedantigenicity associated with glutaraldehyde crosslinking is due tonon-specific cytotoxicity rather than a specific effect on antigenicdeterminants. Glutaraldehyde treatment is an acceptable way to increasedurability and reduce antigenicity of collagenous materials as comparedto those that are noncrosslinked. However, glutaraldehyde crosslinkingcollagen materials significantly limits the body's ability to remodelthe prosthesis (Roe, S. C., et al. (1990), supra).

All of the above problems associated with traditional materials stem, inpart, from the inability of the body to recognize any implant as“inert”. Although biologic in origin, extensive chemical modification ofcollagen tends to render it as “foreign”. To improve the long termperformance of implanted collagenous devices, it is important to retainmany of the properties of the natural collagenous tissue. In this“tissue engineering”0 approach, the prosthesis is designed not as apermanent implant but as a scaffold or template for regeneration orremodeling. Tissue engineering design principles incorporate arequirement for isomorphous tissue replacement, wherein thebiodegradation of the implant matrix occurs at about the same functionalrate of tissue replacement (Yannas, I. V. (1995) Regeneration Templates.pp. 1619–1635. In: Bronzino, J. D. (ed.), The Biomedical EngineeringHandbook, CRC Press, Inc., Boca Raton, Fla.).

When such a prosthesis is implanted, it should immediately serve itsrequisite mechanical and/or biological function as a body part. Theprosthesis should also support appropriate host cellularization byingrowth of mesenchymal cells, and in time, through isomorphous tissuereplacement, be replaced with host tissue, wherein the host tissue is afunctional analog of the original tissue. In order to do this, theimplant must not elicit a significant humoral immune response or beeither cytotoxic or pyrogenic to promote healing and development of theneo-tissue.

Prostheses or prosthetic material derived from isolated collagenmolecules, either in powder form or in a solution, have beeninvestigated for surgical repair or for tissue and organ replacement.The source of collagen used in these prosthetic devices is determinateof the prostheses' form and function. U.S. Pat. No. 4,787,900 to Yannasdetails a process for the creation of prosthetic blood vessels out of acollagenous composite formed, ex vivo, from individual collagenmolecules in either powder or solution form. The collagenous compound isa conglomerate of individual collagen molecules and does not retain anyof the structural characteristics of the tissue from which the collagenwas originally derived. Instead, this collagenous composite is a“tangled mass of collagen fibrils” that is later chemically tailoredinto the desired shape and thickness required for repairing the specificblood vessel.

Prostheses or prosthetic material derived from explanted mammaliantissue have been widely investigated for surgical repair or for tissueand organ replacement. The tissue is typically processed to removecellular components leaving a natural tissue matrix. Further processing,such as crosslinking, disinfecting or forming into shapes have also beeninvestigated. U.S. Pat. No. 3,562,820 to Braun discloses tubular, sheetand strip forms of prostheses formed from submucosa adhered together byuse of a binder paste such as a collagen fiber paste or by use of anacid or alkaline medium. U.S. Pat. No. 4,502,159 to Woodroof provides atubular prosthesis formed from pericardial tissue in which the tissue iscleaned of fat, fibers and extraneous debris and then placed inphosphate buffered saline. The pericardial tissue is then placed on amandrel and the seam is then closed by suture and the tissue is thencrosslinked. U.S. Pat. No. 4,703,108 to Silver provides a biodegradablematrix from soluble collagen solutions or insoluble collagen dispersionswhich are freeze dried and then crosslinked to form a porous collagenmatrix. U.S. Pat. No. 4,776,853 to Klement provides a process forpreparing biological material for implant that includes extracting cellsusing a hypertonic solution at an alkaline pH followed by a high saltsolution containing detergent; subjecting the tissue to protease freeenzyme solution and then an anionic detergent solution. U.S. Pat. No.4,801,299 to Brendel discloses a method of processing body derived wholestructures for implantation by treating the body derived tissue withdetergents to remove cellular structures, nucleic acids, and lipids, toleave an extracellular matrix which is then sterilized beforeimplantation. U.S. Pat. No. 4,902,508 to Badylak discloses a three layertissue graft composition derived from small intestine comprising tunicasubmucosa, the muscularis mucosa, and stratum compactum of the tunicamucosa. The method of obtaining tissue graft composition comprisesabrading the intestinal tissue followed by treatment with an antibioticsolution. U.S. Pat. No. 5,336,616 to Livesey discloses a method ofprocessing biological tissues by treatment of tissue to remove cells,treatment with a cryoprotectant solution, freezing, rehydration, andfinally, innoculation with cells to repopulate the tissue. U.S. Pat. No.4,597,762 to Walter discloses a method of preparing collagenousprostheses through proteolysis, crosslinking with glutaraldehyde,welding and subsequent sterilization of animal hide or other mammaliantissues.

It is a continuing goal of researchers to develop implantable prostheseswhich can successfuily be used to replace or to facilitate the repair ofmammalian tissues, such as abdominal wall defects and vasculature, sothat the intrinsic strength, resillience, and biocompatability of thehost's own cells may be optimally exploited in the repair process.

SUMMARY OF THE INVENTION

The present invention overcomes the difficulties of the materialscurrently available and provides a prosthetic device for use in therepair, augmentation, or replacement of damaged tissues and organs. Thisinvention is directed to a prosthetic material, which, when implantedinto a mammalian host, undergoes controlled biodegradation accompaniedby adequate living cell replacement, or neo-tissue formation, such thatthe original implanted prosthesis is ultimately remodeled and completelyreplaced by host derived tissue and cells. The prosthesis of thisinvention, a material for tissue repair, comprises a non-antigenic,sterile, completely bioremodelable collagenous material derived frommanunalian tissue. The prosthesis of this invention utilizespre-existing, naturally-formed layers of biological collagen forsurgical repair or for tissue and organ replacement. Unlike the tissuerepair fabrics that are currently available, which use collagenouscomposites formed from reconstituted individual collagen molecules, thecollagenous tissue of the present invention retains the structuralcharacteristics of the tissue from which it has been derived. Thiscollagenous tissue of the present invention is able to be layered andbonded together to form multilayer sheets, tubes, or complex shapedprostheses. The bonded collagen layers of the invention are structurallystable, pliable, semi-permeable, and suturable.

Each layer of the prosthetic material of this invention are completelybioremodelable and is replaced by host cells to effectively become ahost tissue. Moreover, because the present invention is comprised ofnaturally-formed pre-existing collagen layers which have been harvestedfrom other mammillian tissues, the risk of a significant humoralresponse has been greatly decreased. It is, therefore, an object of thisinvention to provide a tissue repair fabric that does not exhibit theabove-mentioned shortcomings associated with many of the grafts nowbeing used clinically.

Another object is the provision of a prosthetic material that will allowfor and facilitate tissue ingrowth and/or organ regeneration at the siteof implantation that is a sterile, non-pyrogenic, and non-antigenicmaterial derived from mammalian collagenous tissue. Prostheses preparedfrom this material, when engrafted to a recipient host or patient, donot elicit a significant humoral immune response. Instead, theprostheses is accepted into the recipient host or patient as non-foreignmaterial and the bioremodeling can proceed without interference frompotential immune responses to foreign materials. Prostheses formed fromthe material concomitantly undergoes controlled bioremodeling occurringwith adequate living cell replacement such that the original implantedprosthesis is completely remodeled by the patient's living cells to forma regenerated organ or tissue.

A further object of the current invention is to provide a simple,repeatable method for manufacturing a tissue repair fabric.

Still another object of this invention is to provide a method for use ofa novel multipurpose tissue repair fabric in autografting, allografting,and heterografting indications.

Still a further object is to provide a novel tissue repair fabric thatcan be implanted using conventional surgical techniques.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to tissue engineered prostheses, which, whenimplanted into a mammalian host, can serve as a functioning repair,augmentation, or replacement body part, or tissue structure, and willundergo controlled biodegradation occurring concomitantly withremodeling by the host's cells. The prosthesis of this invention, in itsvarious embodiments, thus has dual properties: First, it functions as asubstitute body part, and second, while still functioning as asubstitute body part, it functions as a remodeling template for theingrowth of host cells. In order to do this, the prosthetic material ofthis invention, a tissue repair fabric, was developed comprisingmammalian derived collagenous tissue that is rendered non-antigenic andis able to be bonded to itself or another. Although the prostheses willbe illustrated through construction of various devices and constructs,the invention is not so limited. It will be appreciated that the devicedesign in its shape and thickness is to be selected depending on theultimate indication for the construct.

In the preferred embodiment, the collagenous material from which to formprostheses, or the prosthesis itself, is rendered sterile,non-pyrogenic, and non-antigenic. The prosthesis, when engrafted to arecipient host or patient, does not elicit a significant humoral immuneresponse. An acceptable level of response is one that demonstrates nosignificant increase in antibody titer to collagenous tissue proteinsfrom baseline titer levels when blood serum obtained from a recipient ofa prosthesis is tested for antibodies to proteins in extracts of thecollagenous tissue.

In the preferred method, the tissue repair material or the prosthesisitself is rendered non-antigenic, while maintaining the ability for theprosthesis to concomitantly undergo controlled bioremodeling occurringwith adequate living cell replacement. The method of preparing anon-antigenic prosthetic collagen material, comprises disinfection ofthe material by a method to prevent microbial degradation of thematerial, preferably by use of a solution comprising peracetic acid; andcrosslinking the disinfected collagen material with a crosslinkingagent, preferably 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC).

Also in the preferred embodiment, collagenous tissues derived from themammalian body are used to make said collagen material. Collagenoustissue sources include, but are not limited to intestine, fascia lata,pericardium, and dura mater. The most preferred material for use is thetunica submucosa layer of the small intestine. The tunica submucosa ispreferably separated, or delaminated, from the other layers of the smallintestine. This layer is referred to hereinafter as the IntestinalCollagen Layer (“ICL”). Further, the collagen layers of the prostheticdevice may be from the same collagen material, such as two or morelayers of ICL, or from different collagen materials, such as one or morelayers of ICL and one or more layers of facia lata.

The submucosa, or the intestinal collagen layer (ICL), from a mammaliansource, typically pigs, cows, or sheep, is mechanically cleaned bysqueezing the raw material between opposing rollers to remove themuscular layers (tunica muscularis) and the mucosa (tunica mucosa). Thetunica submucosa of the small intestine is harder and stiffer than thesurrounding tissue, and the rollers squeeze the softer components fromthe submucosa. In the examples that follow, the ICL was mechanicallyharvested from porcine small intestine using a Bitterling gut cleaningmachine.

As the mechanically cleaned submucosa may have some hidden, visiblynonapparent debris that affects the consistency of the mechanicalproperties, the submucosa may be chemically cleaned to remove debris andother substances, other than collagen, for example, by soaking in buffersolutions at 4° C., or by soaking with NaOH or trypsin, or other knowncleaning techniques. Alternative means employing detergents such asTRITON X-100™ (Rohm and Haas) or sodium dodecylsulfate (SDS); enzymessuch as dispase, trypsin, or thermolysin; and/or chelating agents suchas ethylenediaminetetracetic acid (EDTA) orethylenebis(oxyethylenitrilo)tetracetic acid (EGTA) may also be includedin the chemical cleaning method.

After cleaning, the (ICL) should be decontaminated or disinfected,preferably with the use of dilute peracetic acid solutions as describedin U.S. Pat. No. 5,460,962, incorporated herein by reference.Decontamination or disinfection of the material is done to preventdegradation of the collagenous matrix by bacteria or proteolyticenzymes. Other disinfectant solutions and systems for use with collagenare known in the art and can be used so long as after the disinfectiontreatment there is no interference in the ability of the material to beremodeled.

In a preferred embodiment, the prosthetic device of this invention hastwo or more superimposed collagen layers that are bonded together. Asused herein, “bonded collagen layers” means composed of two or morelayers of the same or different collagen material treated in a mannersuch that the layers are superimposed on each other and are sufficientlyheld together by self-lamination. The bonding of the collagen layers maybe accomplished in a number of different ways: by heat welding orbonding, adhesives, chemical linking, or sutures.

In the preferred method, and in the examples that follow, the ICL isdisinfected with a peracetic acid solution at a concentration betweenabout 0.01 and 0.3% v/v in water, preferably about 0.1%, at aneutralized pH between about pH 6 and pH 8 and stored until use at about4° C. in phosphate buffered saline (PBS). The ICL is cut longitudinallyand flattened onto a solid, flat plate. One or more successive layersare then superimposed onto one another. A second solid flat plate isplaced on top of the layers and the two plates are clamped tightlytogether. The complete apparatus, clamped plates and collagen layers,are then heated for a time and under conditions sufficient to effect thebonding of the collagen layers together. The amount of heat appliedshould be sufficiently high to allow the collagen to bond, but not sohigh as to cause the collagen to irreversibly denature. The time of theheating and bonding will depend upon the type of collagen material layerused, the moisture content and thickness of the material, and theapplied heat. A typical range of heat is from about 50° C. to about 75°C., more typically 60° C. to 65° C. and most typically 62° C. A typicalrange of times will be from about 7 minutes to about 24 hours, typicallyabout one hour. The degree of heat and the amount of time that the heatis applied can be readily ascertained through routine experimentation byvarying the heat and time parameters. The bonding step may beaccomplished in a conventional oven, although other apparatus or heatapplications may be used including, but not limited to, a water bath,laser energy, or electrical heat conduction. Immediately following theheating and bonding, the apparatus is cooled, in air or a water bath, ata range between room temperature at 20° C. and 1° C. Rapid cooling,termed quenching, will immediately, or almost immediately, stop theheating action. To accomplish this step, the apparatus may be cooled,typically in a water bath, with a temperature preferably between about1° C. to about 10° C., most preferably about 4° C. Although coolingtemperatures below 1° C. may be used, care will need to be taken not tofreeze the collagen layers, which may cause structural damage. Inaddition, temperatures above 10° C. may be used in quenching, but if thetemperature of the quench is too high, then the heating may not bestopped in time to sufficiently fix the collagen layers in their currentconfiguration.

The prosthetic material or multi-layered construct is preferably thencrosslinked. Crosslinking the bonded prosthetic device provides strengthand some durability to the device to improve handling properties.Crosslinking agents should be selected so as to produce a biocompatiblematerial capable of being remodeled by host cells. Various types ofcrosslinking agents are known in the art and can be used such as riboseand other sugars, oxidative agents and dehydrothermal (DHT) methods. Apreferred crosslinking agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). The crosslinking solution containingEDC and water may also contain acetone. In a preferred embodiment,sulfo-N-hydroxysuccinimide is added to the crosslinking agent (Staros,J. V., Biochem. 21, 3950–3955, 1982).

In a preferred embodiment, a method comprising disinfection withperacetic acid and subsequent crosslinking with EDC of the ICL materialis performed to reduce the antigenicity of the material. Theimmunoreactive proteins present in non-sterilized, non-crosslinked ICLare either reduced or removed, or their epitopes have been modified suchthat they no longer elicit a significant humoral immune response. Graftimplants of this material do, however, show an initial transientinflammatory response as a result of a wound healing response. As usedherein, the term “non-antigenic” means not eliciting a significanthumoral immune response in a host or patient in whom a prosthesis isimplanted. An acceptable level of response is one that demonstrates nosignificant increase in antibody titer to collagenous tissue proteinsfrom baseline titer levels when blood serum obtained from a recipient ofa prosthesis is tested for antibodies to proteins in extracts of thecollagenous tissue. For a patient or host previously non-sensitized tocollagenous tissue proteins, the preferable serum antibody titer is 1:40or less.

Prostheses of the preferred embodiment are also preferablynon-pyrogenic. A prosthesis that is pyrogenic, when engrafted to arecipient host or patient, will cause a febrile reaction in the patient,thus affecting the ability of the prosthesis to be remodeled. Pyrogensare tested by intravenous injection of a solution containing a sample ofmaterial into three test rabbits. A temperature sensing probe ispositioned in the rectum of the rabbits to monitor temperature changes.If there is a rise in temperature in any rabbit above 0.5° C., then thetest for that sample is continued in five more rabbits. If not more thanthree of the eight rabbits show individual rises in temperature of 0.5°C. or more and the sum of the eight individual maximum temperature risesdoes not exceed 3.3° C., the material under examination meets therequirements for the absence of pyrogens. (Pyrogen Test (151), pp.1718–1719. In: The United States Pharmacopeia (USP) 23 The United StatesPharmacopeial Convention, Inc., Rockville, Md.)

The tissue repair fabric of this invention, functioning as a substitutebody part, may be flat, tubular, or of complex geometry. The shape ofthe tissue repair fabric will be decided by its intended use. Thus, whenforming the bonding layers of the prosthesis of this invention, the moldor plate can be fashioned to accommodate the desired shape. The tissuerepair fabric can be implanted to repair, augment, or replace diseasedor damaged organs, such as abdominal wall defects, pericardium, hernias,and various other organs and structures including, but not limited to,bone, periosteum, perichondrium, intervertebral disc, articularcartilage, dermis, epidermis, bowel, ligaments, and tendons. Inaddition, the tissue repair fabric can be used as a vascular orintra-cardiac patch, or as a replacement heart valve.

Flat sheets may be used, for example, to support prolapsed orhypermobile organs by using the sheet as a sling for the organs. Thissling can support organs such as bladder or uterus.

Tubular grafts may be used, for example, to replace cross sections oftubular organs such as vasculature, esophagus, trachea, intestine, andfallopian tubes. These organs have a basic tubular shape with an outersurface and a lurinal surface.

In addition, flat sheets and tubular structures can be formed togetherto form a complex structure to replace or augment cardiac or venousvalves.

In addition to functioning as a substitute body part or support, thesecond function of the prosthesis is that of a template or scaffold forbioremodeling. “Bioremodeling” is used herein to mean the production ofstructural collagen, vascularization, and epithelialization by theingrowth of host cells at a functional rate about equal to the rate ofbiodegradation of the implanted prosthesis by host cells and enzymes.The tissue repair fabric retains the characteristics of the originallyimplanted prosthesis while it is remodeled by the host into all, orsubstantially all, host tissue, and as such, is functional as an analogof the tissue it repairs or replaces. Thus, each layer of the prosthesisis completely bioremodelable and subsequently replaced by host cells.

The mechanical properties include mechanical integrity such that thetissue repair fabric resists creep during bioremodeling, andadditionally is pliable and suturable. The term “pliable” means goodhandling properties. The term “suturable” means that the mechanicalproperties of the layer include suture retention which permits needlesand suture materials to pass through the prosthesis material at the timeof suturing of the prosthesis to sections of native tissue, a processknown as anastomosis. During suturing, such prostheses must not tear asa result of the tensile forces applied to them by the suture, nor shouldthey tear when the suture is knotted. Suturability of tissue repairfabric, i.e., the ability of prostheses to resist tearing while beingsutured, is related to the intrinsic mechanical strength of theprosthesis material, the thickness of the graft, the tension applied tothe suture, and the rate at which the knot is pulled closed.

As used herein, the term “non-creeping” means that the biomechanicalproperties of the prosthesis impart durability so that the prosthesis isnot stretched, distended, or expanded beyond normal limits afterimplantation. As is described below, total stretch of the implantedprosthesis of this invention is within acceptable limits. The prosthesisof this invention acquires a resistance to stretching as a function ofpost-implantation cellular bioremodeling by replacement of structuralcollagen by host cells at a faster rate than the loss of mechanicalstrength of the implanted materials due from biodegradation andremodeling. The tissue repair fabric of the present invention is“semi-permeable,” even though it has been crosslinked. Semi-permeabilitypermits the ingrowth of host cells for remodeling or for deposition ofthe collagenous layer. The “non-porous” quality of the prosthesisprevents the passage of fluids that are intended to be retained by theimplantation of the prosthesis. Conversely, pores may be formed in theprosthesis if the quality is required for an application of theprosthesis.

The mechanical integrity of the prosthesis of this invention is also inits ability to be draped or folded, as well as the ability to cut ortrim the prosthesis obtaining a clean edge without delaminating orfraying the edges of the construct.

Additionally, in another embodiment of the invention, mechanicallysheared or chopped collagen fibers can be included between the collagenlayers adding bulk to the construct and providing a mechanism for adifferential rate of remodeling by host cells. The properties of theconstruct incorporating the fibers may be altered by variations in thelength and diameter of the fibers; variations on the proportion of thefibers used, and fully or partially crosslinking fibers. The length ofthe fibers can range from 0.1 cm to 5.0 cm.

In another embodiment of the invention, collagen threads, such as thosedisclosed in U.S. Pat. No. 5,378,469 and incorporated herein byreference, can be incorporated into the multilayered tissue repairfabric for reinforcement or for different functional rates ofremodeling. For example, a helix or “twist”, of a braid of 20 to 200denier collagen thread may be applied to the surface of the tissuerepair fabric. The diameter size of the helix or braid of collagenthread can range from 50 to 500 microns, preferably 100 to 200 microns.Thus, the properties of the tissue repair fabric layer can be varied bythe geometry of the thread used for the reinforcement. The functionalityof the design will be dependent on the geometry of the braid or twist.Additionally, collagen thread constructs such as a felt, a flat knittedor woven fabric, or a three-dimensional knitted, woven or braided fabricmay be incorporated between the layers or on the surface of theconstruct. Some embodiments may also include a collagen gel between thelayers alone or with a drug, growth factor or antibiotic to function asa delivery system. Additionally, a collagen gel could be incorporatedwith a thread or a thread construct between the layers.

As will be appreciated by those of skill in the art, many of theembodiments incorporating collagen gel, thread or a thread constructwill also affect the physical properties, such as compliance, radialstrength, kink resistance, suture retention, and pliability. Physicalproperties of the thread or the thread construct may also be varied bycrosslinking the threads.

In some embodiments, additional collagenous layers may be added to theouter or inner surfaces of the bonded collagen layers to create a smoothflow surface for its ultimate application as described in PCTInternational Publication No. WO 95/22301, the contents of which areincorporated herein by reference. This smooth collagenous layer alsopromotes host cell attachment which facilitates ingrowth andbioremodeling. As described in PCT International Publication No. WO95/22301, this smooth collagenous layer may be made from acid-extractedfibrillar or non-fibrillar collagen, which is predominantly type Icollagen, but may also include other types of collagen. The collagenused may be derived from any number of mammalian sources, typicallybovine, porcine, or ovine skin or tendons. The collagen preferably hasbeen processed by acid extraction to result in a fibril dispersion orgel of high purity. Collagen may be acid-extracted from the collagensource using a weak acid, such as acetic, citric, or formic acid. Onceextracted into solution, the collagen can be salt-precipitated usingNaCl and recovered, using standard techniques such as centrifugation orfiltration. Details of acid extracted collagen from bovine tendon aredescribed, for example, in U.S. Pat. No. 5,106,949, incorporated hereinby reference.

Collagen dispersions or gels for use in the present invention aregenerally at a concentration of about 1 to 10 mg/mL, preferably, fromabout 2 to 6 mg/mL, and most preferably at about 3 to 5 mg/mL and at pHof about 2 to 4. A preferred solvent for the collagen is dilute aceticacid, e.g., about 0.05 to 0.1%. Other conventional solvents for collagenmay be used as long as such solvents are compatible.

Once the prosthetic device has been produced, it may be air dried,packaged, and sterilized with gamma irradiation, typically 2.5 Mrad, andstored. Terminal sterilization employing chemical solutions such asperacetic acid solutions as described in U.S. Pat. No. 5,460,962,incorporated herein, may also be used.

In the examples that follow; the ICL is cut longitudinally and flattenedout onto a glass plate, although any inert non-insulated firm mold maybe used. In addition, the mold can be any shape: flat, rounded, orcomplex. In a rounded or complex mold, the bottom and upper mold pieceswill be appropriately constructed so as to form the completed prosthesisinto the desired shape. Once so constructed, the prosthesis will keepits shape. Thus, for example, if the prosthesis is formed into a roundedshape, it can be used as a heart valve leaflet replacement.

The multi-layered tissue repair fabric may be tubulated by variousalternative means or combinations thereof. The multilayered tissuerepair fabric may be formed into a tube in either the normal or theeverted position. The tube may be made mechanically by suturing, usinginterrupted sutures with suitable suture material and is advantageous asit allows the tube to be trimmed and shaped by the surgeon at the timeof implantation without unraveling. Other processes to seam thesubmucosa may include adhesive bonding, such as the use of fibrin-basedglues or industrial-type adhesives such as polyurethane, vinyl acetateor polyepoxy. Preferably heat bonding techniques may also be usedincluding laser welding or heat welding of the seam, followed byquenching, to seal the sides of the thus formed tube. Other mechanicalmeans are possible, such as using pop rivets or staples. With thesetubulation techniques, the ends of the sides may be either butt ended oroverlapped. If the sides are overlapped, the seam may be trimmed oncethe tube is formed. In addition, these tubulation techniques aretypically done on a mandrel so as to determine the desired diameter.

The thus formed structural tube can be kept on a mandrel or othersuitable spindle for further processing. To control functional rates ofbiodegradation and therefore the rate of prosthesis strength decreaseduring bioremodeling, the prosthesis is preferably crosslinked, using asuitable * crosslinking agent, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiumide hydrochloride (EDC). Crosslinking the prosthesis also aidsin preventing luminal creep, in keeping the tube diameter uniform, andin increasing the burst strength. The bond strength of a seam ormultilayer prosthesis is increased when heat or dehydration bondingmethods are used. It is believed that crosslinking the intestinalcollagen layer also improves the suture retention strength by improvingresistance to crack propagation.

Collagen may be deposited on the internal or external surface of the ICLas described in Example 5 of U.S. Pat. No. 5,256,418, incorporatedherein by reference. Briefly, when the tissue repair fabric is to betubulated, the multi-layered fabric is fitted at one end by luerfittings and the collagen dispersion fills the tube. This step may alsobe accomplished as described in the above-referenced patent applicationusing a hydrostatic pressure head. The inner layer of collagen can alsobe deposited by flowing collagen into both ends of the tubesimultaneously. The tube is then placed into a bath of 20% polyethyleneglycol (PEG) in isotonic phosphate buffered saline (PBS), neutral pH.The osmotic gradient between the internal collagen solution and outerPEG solution in combination cause a simultaneous concentration anddeposition of the collagen along the lumen of the internal structurallayer wall. The tube is then removed from the PEG bath, and a glass rodwith a desired diameter of the prosthesis lumen is inserted into thecollagen solution, or alternatively, one end of the prosthesis is closedand air pressure is applied internally to keep the tube lumen open. Theprosthesis is then allowed to dry and subsequently is rehydrated in PBS.The thus formed collagen coating, in the form of a dense fibrillarcollagen, fills slight irregularities in the intestinal structurallayer, thus resulting in a prosthesis with both a smooth flow surfaceand a uniform thickness. The procedure also facilitates the bonding ofthe collagen gel to the intestinal collagen layer. A collagenous layerof varying thickness and density can be produced by changing thedeposition conditions which can be determined by routine parameterchanges. The same procedures can be used to apply the collagen to theouter surface of the ICL to create a three-layer prosthesis.

The prosthesis construct is thrombogenic in small diameter blood vesselreplacements. It can only be used in vascular applications in high flow(large diameter) vessels. Therefore, the prosthesis must be renderednon-thrombogenic if to be useful for small diameter blood vessel repairor replacement.

Heparin can be applied to the prosthesis, by a variety of well-knowntechniques. For illustration, heparin can be applied to the prosthesisin the following three ways. First, benzalkonium heparin (BA-Hep)solution can be applied to the prosthesis by dipping the prosthesis inthe solution and then air-drying it. This procedure treats the collagenwith an ionically bound BA-Hep complex. Second, EDC can be used toactivate the heparin, then to covalently bond the heparin to thecollagen fiber. Third, EDC can be used to activate the collagen, thencovalently bond protamine to the collagen and then ionically bondheparin to the protamine. Many other coating, bonding, and attachmentprocedures are well known in the art which could also be used.

Treatment of the tissue repair fabric with drugs in addition to or insubstitution for heparin may be accomplished. The drugs may include forexample, growth factors to promote vascularization andepithelialization, such as macrophage derived growth factor (MDGF),platelet derived growth factor (PDGF), endothelial cell derived growthfactor (ECDGF); antibiotics to fight any potential infection from thesurgery implant; or nerve growth factors incorporated into the innercollagenous layer when the prosthesis is used as a conduit for nerveregeneration. In addition to or in substitution for drugs, matrixcomponents such as proteoglycans or glycoproteins or glycosaminoglycansmay be included within the construct.

The tissue repair fabric can be laser drilled to create micron sizedpores, through the completed prosthesis for aid in cell ingrowth usingan excimer laser (e.g. at KrF or ArF wavelengths). The pore size canvary from 10 to 500 microns, but is preferably from about 15 to 50microns and spacing can vary, but about 500 microns on center ispreferred. The tissue repair fabric can be laser drilled at any timeduring the process to make the prosthesis, but is preferably done beforedecontamination or sterilization.

Voids or spaces can also be formed by the method of phase inversion. Atthe time of layering the ICL, between layers is distributed crystallineparticles that are insoluble in the liquid heat source for bonding butshould be soluble in the quench bath or the crosslinking solution. Iflaser or dry heat is used to bond the layers then any solublecrystalline solid may be used as long as it is soluble in the quenchbath or the crosslinking solution. When the crystalline solid issolubilized and has diffused out, there remains a space in-which thesolid had occupied. The size of the particles may vary from 10 to 100microns, but is preferably from about 15 to 50 microns and spacing canvary between particles when distributed between the layers. The numberand size of the voids formed will also affect the physical properties(i.e., compliance, kink resistance, suture retention, pliability).

The following examples are provided to better elucidate the practice ofthe present invention and should not be interpreted in any way to limitthe scope of the present invention. Those skilled in the art willrecognize that various modifications, can be made to the methodsdescribed herein while not departing from the spirit and scope of thepresent invention.

EXAMPLES Example 1 Harvesting and Processing of the Intestinal CollagenLayer from Porcine Intestine

The small intestine of a pig was harvested and mechanically stripped,using a Bitterling gut cleaning machine (Nottingham, UK) which forciblyremoves the fat, muscle and mucosal layers from the tunica submucosausing a combination of mechanical action and washing using hot water.The mechanical action can be described as a series of rollers thatcompress and strip away the successive layers from the tunica submucosawhen the intact intestine is run between them. The tunica submucosa ofthe small intestine is harder and stiffer than the surrounding tissue,and the rollers squeeze the softer components from the submucosa. Theresult of the machine cleaning was such that the submucosal layer of theintestine solely remained. Finally, the submucosa was decontaminatedwith 0.3% peracetic acid for 18 hours at 4° C. and then washed inphosphate buffered saline. The product that remained was an intestinalcollagen layer (ICL).

Example 2 Various Welding Temperatures and EDC Concentrations of ICL

The effects of welding temperature (followed by quenching), weld time,1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) concentration,acetone concentration and crosslinking time, after welding on weldstrength were examined for the ICL two layered tube application. ICL wasporcine derived as described in the Example 1. Strength qualities weremeasured using a suture retention test and a ultimate tensile strength(UTS) test.

ICL was inverted and stretched over a pair of mandrels which wereinserted into an ICL mounting frame. Mandrels were of stainless steeltubing with an external diameter of 4.75 mm. The ICL and mandrels werethen placed in a dehydration chamber set at 20% relative humidity at 4°C. for about 60 minutes. After dehydration, the ICL was removed from thechamber and the mandrels. The lymphatic tag areas were removed and theICL was manually wrapped around the mandrel twice to form an ‘unwelded’bilayer construct. The wrapped ICL was returned to the dehydrationchamber and allowed to dry for another 90 minutes still at 20% relativehumidity to about 50% moisture +/−10%. To determine the amount ofmoisture present in a sample construct, a CEM™ oven was used.

A THERMOCENTER™ oven was set for the designated temperature treatmentfor the constructs to be welded. Temperatures tested for welding rangedfrom 55° to 70° C. Once the constructs were placed in the oven, the ovenwas allowed to equilibrate before timing began. The constructs wereallowed to remain in the chamber for the time required for thatcondition. Welding times ranged from 7 to 30 minutes. As soon as thetime was completed the constructs were removed from the chamber andplaced in a 4° C. water bath for about 2 to 5 minutes. The weldedconstructs were then returned to the dehydration chamber for about 30minutes until dehydrated to about 20% +/−10%.

After dehydration, constructs were inserted into a vessel containing EDCin either deionized water or deionized water and acetone at theconcentrations appropriate for the conditions tested. EDC concentrationstested were 50, 100, and 200 mM. Acetone concentrations tested were 0,50, and 90% in water. The time duration for crosslinking was determinedby the conditions tested. Crosslinking times were 6, 12, and 24 hours.After crosslinking, the construct was removed from the solution andrinsed with physiological pH phosphate buffered saline (PBS) three timesat room temperature. The welded and crosslinked construct was thenremoved from the mandrel and stored in PBS until testing. In addition tothe thirty constructs that were prepared, two other bilayer constructswere prepared by welding at 62° C. for 15 minutes and crosslinked in 100mM EDC in 100% H₂O for 18 hours.

The suture retention test was used to determine the ability of aconstruct to hold a suture. A piece of construct was secured in aCHATTILION™ force measurement device and 1–2 mm bite was taken with aSURGLENE™ 6–0 suture, pulled through one wall of the construct andsecured. The device then pulls at the suture to determine the forcerequired to tear the construct material. The average suture breaksbetween 400–500 g of force; the surgeons pull tends to be 150 g offorce.

The weld/material strength test was performed to determine the UTS of aconstruct. Sample rings of 5 mm lengths were excised from each tube andeach was tested for their ultimate tensile strength (UTS) test using amechanical testing system MTS™. Three sample rings were excised fromeach tube for three test pulls done for each construct for a total of 90pulls. A ring was placed in the grips of the MTS™ and is pulled at arate of 0.02 kg_(force)/sec until the weld slips or breaks, or until thematerial (rather than the weld) breaks.

Example 3 Various Welding Temperatures of ICL

The effect of welding temperature and quenching after welding on weldstrength were examined for the ICL two layered tube application.

An ICL sample of 10 feet long was cut along its length and prepared asin the procedure outlined in Example 2. Six 6 mm diameter tubes rangingbetween 15–20 cm in length were prepared for each temperature condition.

Tubes were subjected to a temperature condition while wet for 3.5 hours.Temperatures conditions were: Room temperature (20° C.), 55° C., 62° C.and 62° C. then immediately quenched in 4° C. bath for one minute. Alltubes were then crosslinked in EDC. Six tubes were placed together in300 mL 100 mM EDC overnight at room temperature. Tubes were then rinsedwith physiological strength phosphate buffered saline aftercrosslinking.

Sample rings of 5 mm lengths were excised from each tube and each wastested for ultimate tensile strength (UTS) test using a MTS™. Fivesample rings were taken from each tube for 5 test pulls on each of 6tubes per condition for a total of 30 pulls.

Weld strength was less consistent for tubes bonded by dehydration atroom temperature as compared to the other temperature treatments whentested using the UTS test. One of the six tubes welded at roomtemperature had UTS measurements comparable to those of the othertreatments. For the tubes welded at other temperatures either with orwithout quenching, there were no differences in weld strength. After UTStesting, it was determined that the breaking of the material was not aseparation of the weld but a material failure in all instances.

Example 4 The Antigenicity of Crosslinked Intestinal Collagen Layer

Fresh samples of porcine submucosal intestinal layer were obtained afterthe cleaning step as described in example 1. Samples were then leftuntreated and stored in water, soaked in physiological strengthphosphate buffered saline, treated with 0.1% peracetic acid, or weretreated with 0.1% peracetic acid and then crosslinked with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).Samples were then extracted with a solution of 0.5 M NaCl/0.1 M tartaricacid for about 18 hours.

Two 12% Tris-glycine sodium dodecylsulfate-polyacrylamide gels (NovexPrecast Gels cat# EC6009) were run and then transferred after about 18hours to 0.45 μ nitrocellulose paper. Tartaric acid extracts of eitheruntreated or treated ICL were run against a control standard lanecontaining: 10 μd Kaleidoscope Prestained Standards (Bio-Rad cat#161-0324): 2 μl biotinylated SDS-PAGE low range molecular weightstandards (Bio-Rad cat# 161-0306): 6 μl loading buffer; 10 μd of controlstandard were loaded to each lane. The gel was blotted for about 2 hourswith 1% dry non-fat milk (Carnation) in phosphate buffered saline. Thegel was then washed three times with borate buffered saline/Tween with200 μl of wash per lane. Primary antibody in 200 μl of Rb serum andborate buffered saline (100 mM boric acid: 25 mM sodium borate: 150 mMNaCl)Tween was added to each lane at various titer range (1:40, 1:160,1:640 and 1:2560). The gel was then incubated at room temperature forone hour on a rocker platform (Bellco Biotechnology) with the speed setat 10. The gel was then washed again three times with borate bufferedsaline/Tween. Secondary antibody, goat anti-rabbit Ig-AP (SouthernBiotechnology Associates Inc. cat# 4010-04) at a 1:1000 dilution wasadded to the lanes at 200 μl per lane and the gel was incubated for onehour at room temperature on a rocker platform. The nitrocellulosemembrane was then immersed in AP color development solution whileincubated at room temperature on a rocker platform until colordevelopment was complete. Development was stopped by washing themembrane in deionized water for ten minutes on a rocker platform whilechanging the water once during the ten minutes. The membrane was thenair dried.

The results obtained from analysis of the gel suggest that theantigenicity of the porcine derived ICL treated with peracetic acid andEDC has greatly reduced antigenicity as compared to the othertreatments.

Example 5 Six Layered Tissue Repair Fabric as an Abdominal Wall Patch

Six layers of porcine intestinal collagen were superimposed onto oneanother on a glass plate. A second plate of glass was then placed on topof the intestinal collagen layers and clamped tightly to the firstplate. The apparatus was placed into a conventional type oven at 62° C.for one hour. Immediately following heating, the apparatus was placedinto a 4° C. water bath for ten minutes. The apparatus was disassembled,the intestinal collagen layers removed, and treated with 100 mM1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in50% acetone for four hours at 25° C. The material was bagged andsterlized by gamma irradiation (2.5 Mrad).

The tissue repair fabric was sutured in a 3 cm×5 cm defect in themidline of New Zealand White rabbits (4 kg) using a continuous 2–0prolene suture. Animals were sacrificed at four weeks, ten weeks, and 16weeks, and examined grossly, mechanically, and histologically. Grossexamination showed minimal inflammation and swelling. The graft wascovered with a glistening tissue layer which appeared to be continuouswith the parietal peritoneum. Small blood vessels could be seenproceeding circumferentially from the periphery to the center of thepatch. Mechanically the graft was stable with no reherniation observed.Histological examination revealed relatively few inflammatory cells andthose that were observed were primarily near the margin of the graft(due to the presence of prolene suture material). The peritoneal surfacewas smooth and covered entirely by mesothelium.

Example 6 Two Layered Tissue Repair Fabric as a Pericardial Repair Patch

Two layers of porcine intestinal collagen were superimposed onto oneanother on a glass plate. A second plate of glass was then placed on topof the intestinal collagen layers and clamped tightly to the firstplate. The apparatus was placed into a conventional type oven at 62° C.for one hour. Immediately following heating, the apparatus was placedinto a 4° C. water bath for ten minutes. The apparatus was disassembled,the intestinal collagen layers removed, and treated with 10 mM1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in50% acetone for four hours at 25° C. The material was bagged andsterilized by gamma irradiation (2.5 Mrad).

A 3×3 cm portion of New Zealand white rabbit pericardium was excised andreplaced with a same size piece of tissue repair fabric (anastomosedwith interrupted sutures of 7–0 prolene). Animals were sacrificed atfour weeks and at 180 days, examined grossly, mechanically, andhistologically. Gross examination showed minimal inflammation andswelling. Small blood vessels could be seen proceeding circumferentiallyfrom the periphery to the center to the graft. Mechanically, the graftwas stable without adhesion to either the sternum or pericardial tissue.Histological examination revealed relatively few inflammatory cells andthose that were observed were primarily near the margin of the graft(due to the presence of prolene suture material).

Example 7 Hernia Repair Device

A prototype device for hernia repair was developed using ICL to have ahollow inner region. The device, when completed, had a roundconformation bonded at the periphery and a swollen inner region renderedswollen by the inclusion of physiological strength phosphate bufferedsaline. The inner region can optionally accommodate a wire coil foradded rigidity or other substance for structural support or delivery ofsubstance.

To assemble ICL multilayer sheets, 15 cm lengths of ICL were trimrned oflymphatic tags and cut down the side with the tags to form a sheet.Sheets were patted dry with Texwipes. On a clean glass plate (6″×8″),sheets were layered mucosal side down. In this case, two two-layer andtwo four-layer patches were constructed by layering either two or fourlayers of ICL on the glass plates. A second glass plate (6″×8″) wasplaced on top of the last ICL layer and the plates were clamped togetherand then placed in a hydrated oven at 62° C. for one hour. Constructswere then quenched in deionized water at 4° C. for about ten minutes.The glass plates were then removed from the bath and a plate removedfrom each patch. The now bonded ICL layers were then smoothed out toremove any creases or bubbles. The glass plate was replaced upon the ICLlayers and returned to the hydrated oven for 30–60 minutes until dry.Patches were removed from the oven and partially rehydrated by sprayingwith physiological strength phosphate buffered saline.

For the construction of a bi-layer construct, one bi-layer patch wasremoved from the glass plates and placed upon the other bi-layer patchstill on the other glass plate. An annular plate (d_(out)=8.75 cm;d_(in)=6 cm) was placed upon the second patch. About 10 cc ofphysiological strength phosphate buffered saline was then injectedthrough a 25 gauge needle between the two bilayer patches. A secondglass plate was then placed on top of the annular plate and were thenclamped together. For the construction of a four-layer construct, thesame steps were followed except that two four-layer patches were usedrather than two bi-layer patches. The constructs were placed in ahydrated oven at 62° C. for one hour. Constructs were then quenched indeionized water at 4° C. for about fifteen minutes. Constructs were thencrosslinked in 200 mL 100 mM EDC in 50% acetone for about 18 hours andthen rinsed with deionized water. The constructs were then trimmed toshape with a razor blade to the size of the outer edge of the annularplate.

Example 8 Intervertebral Disc Replacement

ICL, dense fibrillar collagen and hyaluronic acid were configured toclosely approximate the anatomic structure and composition of anintervertebral disc.

Dense fibrillar collagen diskettes containing hyaluronic acid wereprepared. 9 mg hyaluronic acid sodium salt derived from bovine trachea(Sigma) was dissolved in 3 mL 0.5 N acetic acid. 15 mL of 5 mg/mLcollagen (Antek) was added and mixed. The mixture was centrifuged toremove air bubbles. To ,three transwells (Costar) in a six well plate(Costar) was added 5 mL of the solution. To the area outside thetranswell was added N600 PEG to cover the bottom of the membranes. Theplate was maintained at 4° C. on an orbital shaker table at low speedfor about 22 hours with one exchange of PEG solution after 5.5 hours.PEG solution was removed and the transwells dehydrated at 4° C./20% Rhovernight.

To assemble ICL multilayer sheets, 15 cm lengths of ICL were trimmed oflymph tags and cut down the side with the tags to form a sheet. Sheetswere patted dry with Texwipes. On a clean glass plate, sheets werelayered mucosal side down to five layers thick and a second glass platewas laid on top of the fifth layer. Five five-layer patches wereconstructed. The plates with the ICL between were clamped together andplaced in a hydrated oven at 62° C. for one hour. Constructs were thenquenched in RODI water at 4° C. for about ten minutes then were removedform the quench bath and stored at 4° C. until assembly of the disc.

To another glass plate, one large patch was laid. A slightly smallerpatch was laid upon the first patch aligned to one edge of the largerpatch. One patch was cut in half and a hole was cut in the center ofeach approximating the size of the DFC diskettes. With the center holesaligned, the two halves were laid upon the second patch aligned to thesame edge. Three rehydrated DFC/HA diskettes were placed within thecenter hole. Another slightly smaller patch was laid upon the two halvescontaining the DFC diskettes and a larger patch laid upon the smallerpatch, both aligned to the same edge. A second glass plate was placed ontop of the construct. The resultant shape was that of a wedge with thethicker side being the one with the aligned edges tapering to theopposite side. The thus formed device was placed in a hydrated oven at62° C. for one hour and then quenched in RODI water at 4° C. for aboutten minutes. The device was then crosslinked in 100 mM EDC (Sigma) in90% acetone (Baxter) for about five hours and then rinsed with threeexchanges of phosphate buffered saline. The edges of the device werethen trimmed with a razor blade.

Example 9 The Formation of Vascular Graft Construct

The proximal jejunum of a pig was harvested and processed with a GutCleaning Machine (Bitterling, Nottingham, UK) and then decontaminatedwith peracetic acid solution as described in example 1. The peraceticacid treated ICL (PA-ICL) was cut open longitudinally and lymphatic tagareas were removed to form a sheet of ICL. The ICL sheets were wrappedaround a 6.0 mm diameter stainless steel mandrels to form bilayerconstructs. The constructs (on mandrels) were then placed in anequilibrated THERMOCENTER™ oven chamber set at 62° C. for about 1 hour.The constructs were removed from the chamber and placed in a 4° C. waterbath for about 2 to 5 minutes. The constructs were chemicallycrosslinked in 50 mL of 100 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in 50/50 water/acetone solution for 18 hours to formperacetic acid treated, EDC crosslinked (PA/EDC-ICL) vascular graftconstructs. The constructs were removed from the mandrels and rinsedwith water to remove residual EDC solution.

After removal from the mandrels, a layer (approximately 200 μm thick) oftype I collagen extracted from bovine tendon, was deposited on theluminal surface of the constructs according to the method described inU.S. Pat. No. 5,256,418, incorporated herein. Polycarbonate barbs (luerlock fittings that are conically shaped on one end) were sealably fixedat either end of the constructs and the constructs were placedhorizontally in a deposition fixture. A 50 mL reservoir of 2.5 mg/mLacid-extracted collagen, prepared by the method described in U.S. Pat.No. 5,106,949, incorporated herein, was attached via the barbs. Thecollagen was allowed to fill the lumen of the ICL tube and was thenplaced into a stirring bath of 20% MW 8000 polyethylene glycol (SigmaChemicals Co.) for 16 hours at 4° C. The apparatus was then dismantledand a 4 mm diameter glass rod was placed into the collagen-filled ICLtube to fix the luminal diameter. The prosthesis was then allowed todry.

The luminal DFC layer was coated with benzalkonium chloride heparin(HBAC) by dipping the grafts three times into an 800 U/mL solution ofHBAC and allowed to dry. Finally, the graft received a final chemicalsterilization treatment in 0.1% v/v peracetic acid. The graft was storedin a dry state until the implant procedure.

Example 10 Implant Studies on Animal Models

Twenty-five mongrel dogs weighing 15–25 kg were fasted overnight andthen anesthetized with intravenous thiopental (30 mg/kg), entubated, andmaintained with halothane and nitrous oxide. Cefazolin (1000 mg) wasadministered intravenously preoperatively as well as postoperatively.Each dog received either an aortic bypass grafts or a femoralinterposition graft. For the aortic bypass grafts, a midline abdominalincision was made and the aorta exposed from the renal arteries to thebifurcation, followed by the administration of intravenous heparin (100U/kg). The grafts (6 mm×8 cm) were, placed between the distal infrarenalaorta (end-to-side anastomosis) and the aorta just proximal to thebifurcations (end-to-side anastomosis). The aorta was ligated distal tothe proximal anastomosis. The incisions were closed and the dogsmaintained on aspirin for 30 days post surgery. For the femoralinterposition grafts, the animals were opened bilaterally, the femoralarteries exposed, and a 5 cm length excised. The grafts (4 mm×5 cm) wereanastomosed in end-to-end fashion to the femoral artery. On thecontralateral side, a control graft was placed. The incisions wereclosed and the animals were maintained on aspirin for 30 days postsurgery. Post operative follow-up ranged from 30 days to 360 days.Pre-implant, and four and eight weeks post-implant bloods werecollected. Animals were sacrificed at various time points (30 days, 60days, 90 days, 180 days, and 360 days).

New Zealand White rabbits weighing 3.5–4.5 kg were fasted overnight, andthen anesthetized with acepromazine (20 mg) and ketamine (40 mg),entubated, and maintained with ketamine (50 mg/mL), injectedintravenously as needed. Penicillin (60,000 U) was administeredintramuscularly preoperatively. A midline abdominal incision was madeand the aorta exposed from the renal arteries to the bifurcation,followed by the administration of intravenous heparin (100 U/kg). A 3 cmlength of aorta was excised, and the grafts (2.5 mm×3 cm) wereanastomosed in end-to-end fashion to the aorta. The incisions wereclosed and the animals were maintained with no anticoagulant therapypost surgery. Post operative follow-up ranged from 30 days to 360 days.Animals were sacrificed at various time points (30 days, 60 days, 90days, 180 days, and 360 days).

The implants along with adjacent vascular tissues obtained fromsacrificed animals were fixed for transmission electron microscopy (TEM)analysis for 4 hr in a solution of 2.0% paraformaldehyde, 2.5%glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4. Samples were thenpost-fixed in 1.0% OsO4 (in 0.1M sodium cacodylate) and stained en blocwith 2.0% uranyl acetate (aqueous). After secondary fixation allspecimens were dehydrated in a graded ethanol series and propylene oxideand embedded in Epox 812 (Ernest F. Fullam, Rochester, N.Y., USA).Ultrathin (˜700 nm) sections were stained with uranyl acetate and leadcitrate. Sections were examined on a JEOL Instruments JEM100S at 80 kV.For scanning electron microscopy (SEM), samples were fixed for 18 hr inhalf strength Karnovsky's solution and rinsed five times in Sorensen'sphosphate buffer prior to post fixation in 1.0% OsO4 for 1 hr. Sampleswere then rinsed twice in Sorensen's phosphate buffer and three times indouble distilled water. Dehydration was accomplished through an ethanolseries (50%, 70%, 90%, and 100%), followed by critical point drying.Samples were mounted and coated with 60/40 gold/palladium.

ICL graft explants from dogs and rabbits were examined histologically toevaluate host cell ingrowth. Masson's trichrome staining of a 60 dayexplant showed significant host infiltrate. The darker blue stainingshowed collagen of the ICL while matrix surrounding the myofibroblasts,stained lighter blue, showed an abundance of host collagen. High powermagnification of the section showed numerous cells intermingled withinthe ICL. The inflammatory response seen at 30 days had been resolved andthe cellular response was predominantly myofibroblastic. The surface ofthe remodeled graft was lined by endothelial cells as demonstrated bySEM and Factor VIII staining. By 360 days, a mature ‘neo-artery’ hadbeen formed. The neo-adventitia was composed of host collagen bundlespopulated by fibroblast-like cells. The cells and matrices of theremodeled construct appeared quite mature and tissue-like.

Example 11 Generation of Anti-ICL Antibodies

Fresh samples of porcine submucosal intestinal layer were obtained afterthe cleaning step as described in example 1 but were not peracetic acidtreated. Samples were then left untreated (NC-ICL), treated with 0.1%peracetic acid (PA-ICL), or treated with 0.1% peracetic acid and thencrosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (PA/EDC-ICL).

New Zealand White rabbits were immunized with 0.5 mg of any one of thethree types of ICL samples (NC-ICL, PA-ICL, or PA/EDC-ICL) to generateanti-serum. Initially, rabbits were injected subcutaneously with 0.5 mLof homogenized untreated ICL ini Freund's complete adjuvant (1:1, 1mg/mL). Sham rabbits received 0.5 mL of phosphate buffered saline inFreund's complete adjuvant. Rabbits were boosted every 3 to 4 monthswith 0.5 mL of the appropriate form of ICL in Freund's incompleteadjuvant (0.25 mg/mL). Sera were collected 10–14 days after each boost.

Example 12 Generation of ICL Extracts and Characterization ofPotentially Antigenic Proteins Associated with Native Collagen

Proteins were extracted from NC-ICL, PA-ICL, or PA/EDC-ICL usingtartaric acid (Bellon, G., et al (1988) Anal. Biochem. 175: 263–273) orTRITON X-100 (Rohm and Haas). Pulverized NC-ICL, PA-ICL, or PA/EDC-ICL(10% w/v) were mixed with either tartaric acid (0.1 M tartaric acid, 0.5M NaCl) or TRITON X-100 (Rohm and Haas) extraction buffer (TEB; 1%TRITON X-100 in 20 mM Tris HCl (pH 7.2), 2 mM EGTA, 2 mM EDTA, 1 mMphenylmethylsulfonyl fluoride, and 25 mg/mL each of aprotinin,leupeptin, and pepstatin (Sigma, St. Louis, Mo.)). The mixtures wereincubated overnight at 4° C. The extracts were gauze filtered to removedebris, dialyzed against PBS and concentrated using Centriprep-30(Amicon, Danvers, Mass.). Extracts were stored at −80° C. until used.

Tartaric acid and TEB extracts were separated on 10% polyacrylamide gelsby SDS-PAGE according to Laemnili (Laemnili, U.K. (1970) Nature 227:680–685). Gels were either silver stained (Bio-Rad, Hercules, CA) ortransferred to nitrocellulose membranes (Amersham, Arlington Heights,Ill.). Multiple protein bands were visualized in the NC-ICL extracts bysilver staining. In contrast, only two bands were visible in the PA-ICLextracts and no protein bands were seen in the lanes containingPAIEDC-ICL. These results suggest that peracetic acid and EDC treatment,in combination, leads to a decrease in the extractable non-collagenousproteins in ICL.

Immunoblot transfer was done overnight using a Trans-Blot Cell (Bio-Rad)in Tris-Glycine 20% methanol transfer buffer. Nitrocellulose membranescontaining ICL transferred proteins were blocked with Blotto buffer (1%non-fat dry milk in borate buffered saline with 0.1% Tween-20(BBS/Tween)) for one hour at room temperature. The nitrocellulosemembranes were transferred to a multiscreen apparatus containing 12individual lanes. The membranes were washed three times with BBS/Tween.Positive control or test sera (100 μL/lane) were added to the membraneand rocked at room temperature for 1 hour. Each lane was washed threetimes with BBS/Tween. Secondary antibodies: ALPH-labeled goatanti-rabbit Ig or ALPH-labeled goat anti-dog Ig (Southern Biotechnology)were added to the appropriate lanes (100 μL/lane) and streptavidin-AP(100 μL) was added to one of the lanes containing the Kaleidoscopemolecular weight standards (Bio-Rad). An alkaline phosphatase conjugatesubstrate kit (Bio-Rad) was used to visualize the immunoblots.

Rabbit anti-NC-ICL serum, generated by repeated immunization withNC-ICL, was used to detect potentially immunoreactive proteins. Serafrom immunized rabbits recognized antigens with molecular weights in therange of <30, 40–70, and >100 kDa in the tartaric acid extract. Thesesame sera were tested on immunoblots of TEB extracts from NC-ICL.Immunoreactive proteins were detected with molecular weights rangessimilar to those detected in the tartaric acid extract, with additionalreactivity detected in the 70–100 kDa range. The results indicated thatNC-ICL contains multiple proteins which are immunoreactive and theseproteins can be extracted by tartaric acid or TEB. The greater number ofimmunoreactive proteins present in the TEB extract correlated with theincrease in proteins extracted using TEB as compared to tartaric acid.

Example 13 Effect of PA or EDC Treatment of ICL on the Antigenicity ofType I Collagen in ICL

Sera from rabbits immunized with NC-ICL, PA-ICL, or PA/EDC-ICL (seraprepared as described in example 11) or acid extracted type I collagen(Organogenesis, Canton, Mass.) were tested for type I collagen specificantibodies by ELISA. ELISA plates (Immulon II, NUNC, Bridgeport, N.J.)were coated with 200 mL/well of 1 mg/mL acid extracted type I collagenin 0.05 M carbonate buffer (pH 9.6) overnight at 4° C. Plates werewashed twice with PBS/Tween-20 (0.1%). Serum samples from animals orrabbit anti-collagen type I antibody (Southern Biotechnology,Birmingham, Ala.) were added to wells (100 mL/well) and incubated for 1hr at room temperature. Plates were washed three times with PBS/Tween.Secondary antibodies: ALPH-labeled goat anti-rabbit Ig or ALPH-labeledgoat anti-dog Ig (Southern Biotechnology) were added to the appropriatewells and incubated at room temperature for 1 hour. Plates were washedthree times with PBS/Tween. P-nitrophenylphosphate (PNPP) substrate (1mg/mL) was added to each well (100 mL/well). Absorbance was read at 405nm on a SpectraMax microplate reader (Molecular Devices, Sunnydale,Calif.).

Anti-collagen type I antibodies could not be detected in sera fromrabbits immunized with any form of ICL, even at a 1:40 serum dilution.In contrast, rabbits immunized with purified type I collagen hadantibody titer of 1:2560. These data suggest that crosslinking is notnecessary to reduce the antigenicity to collagen type I, since rabbitsimmunized with NC-ICL did not generate anti-collagen type I antibodies.These data thus suggest that the immunodominant proteins in NC-ICL arenon-collagenous proteins. Also, the effect of PA and EDC on reducing theantigenicity of ICL is directed toward the non-collagenous proteins.

Example 14 Effects of Disinfecting and Crosslinking on Antigenicity ofICL

The effect of PA and EDC treatment on the antigenicity of ICL wasdetermined by using anti-NC-ICL antiserum to probe for immunoreactiveproteins present in tartaric acid or TEB extracts of PA or PA/EDCtreated ICL.

Tartaric acid extracts of PA-ICL and TEB extracts of PA/EDC-ICL wereseparated on 10% SDS-PAGE gels and transferred to nitrocellulosemembranes for immunoblot analysis, as described in Example 12. NC-ICLspecific antisera were used to probe for immunoreactive proteins in eachextract. Even when immunoblots of PA-ICL. and PA/EDC-ICL wereoverexposed, no reactivity could be detected in lanes containinganti-NC-ICL antibodies thus suggesting that the immunoreactive proteinsdetected in the NC-ICL are either missing or their epitopes have beenmodified such that they are no longer recognized by anti-NC-ICLanti-serum. To address this latter issue, serum from rabbits immunizedwith either PA-ICL or PA/EDC-ICL was also tested. No antibody bindingwas detected in any of the lanes above background. These data indicatethat even when rabbits were immunized with modified ICL they did notgenerate antibodies which could recognize modified ICL extractedproteins. These results suggest that the proteins removed or modifiedduring the process of disinfecting and crosslinking are the sameproteins responsible for the antigenicity of NC-ICL.

Antibody response of PA-ICL or PA/EDC-ICL immunized rabbits was analyzedby immunoblotting, as described in Example 12. This approach was takento ensure that the lack of reactivity of anti-NC-ICL sera withPA/EDC-ICL was due to the absence of proteins in ICL and not due to aninability to extract proteins which might be accessible to the immunesystem in vivo since crosslinking of collagenous materials with EDCcould reduce the quantity and quality of protein extracted from ICL.Anti-ICL antisera was generated using PA-ICL or PA/EDC-ICL to immunizerabbits. Sera from these rabbits were tested for antibodies specific forproteins in either tartaric acid or TEB protein extracts of NC-ICL.Anti-PA-ICL recognized the 207, 170, and 38–24 kDa proteins recognizedby anti-NC-ICL, but lost reactivity to the lower molecular weightproteins. No bands were detected by the anti-PA/EDC-ICL serum from 1rabbit. Serum from another anti-PAIEDC-ICL rabbit reacted with the 24–38kDa proteins. These data suggested that both PA-ICL and PA/EDC-ICL areless antigenic than NC-ICL. Either the antigenic epitopes of ICL areremoved during the disinfecting and crosslinking process or they aremodified to reduce their antigenicity. In either case, disinfection andcrosslinking resulted in a material whose antigenicity was significantlyreduced.

Example 15 Determination of Humoral Immune Response in Graft Recipients

Dogs were tested for a humoral immune response to ICL graft componentsto determine if ICL must retain its antigenicity to stimulate cellingrowth into the graft. Pre-implant, and four and eight weekspost-implant blood samples were collected from fifteen dogs thatreceived PAIEDC-ICL vascular grafts. Serum from each blood sample wastested for antibodies to proteins in both the tartaric acid and TEBextracts of NC-ICL. Even at a 1:40 dilution of serum, none of the dogstested had antibodies which reacted with ICL proteins. These same serumsamples were tested for the presence of anti-collagen type I antibodiesby ELISA. All serum samples were negative for antibodies to type Icollagen at a serum dilution of 1:40. Masson's trichrome staining ofexplant paraffin sections from these dogs did shown infiltration of hostcells. These results demonstrate that PA/EDC-ICL does not elicit anantibody response when the host is actively remodeling the material.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious to one of skill in the art thatcertain changes and modifications may be practiced within the scope ofthe appended claims.

1. A prosthesis comprising two or more superimposed, bonded layers ofcollagenous tissue which have been crosslinked with a crosslinking agentthat permits bioremodeling, wherein said collagenous tissue is derivedfrom tunica submucosa of the small intestine separated from the otherlayers of the small intestine, wherein the prosthesis is non-antigenic,wherein the prosthesis is sterilized, wherein the two or more layers ofthe prosthesis are completely bioremodelable, and which, when implantedinto a mammalian patient, undergoes controlled biodegradation occurringwith adequate living cell replacement such that the original implantedprosthesis is remodeled by the patient's living cells.
 2. The prosthesisof claim 1 wherein the shape of said prosthesis is flat, tubular, orcomplex.
 3. The prosthesis of claim 1 wherein said collagenous layersare bonded together by heat welding for a time and under conditionssufficient to effect the bonding of the collagenous tissue layers. 4.The prosthesis of claim 1 wherein said prosthesis is crosslinked withthe crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride.
 5. The prosthesis of claim 4 whereinsulfo-N-hydroxysuccinimide is added to the crosslinking agent.
 6. Theprosthesis of claim 4 wherein acetone is added to the crosslinkingagent.
 7. The prosthesis of claim 1 wherein the prosthesis is sterilizedwith peracetic acid.
 8. The prosthesis of claim 1, wherein theprosthesis has one or more surfaces and wherein the one or more surfacesof said prosthesis is coated with a collagenous material which acts as asmooth flow surface.
 9. The prosthesis of claim 1 wherein saidprosthesis further contains pores.
 10. The prosthesis of claim 1 whereinsaid prosthesis is further composed of chopped collagen fibers.
 11. Theprosthesis of claim 1 wherein said prosthesis is further composed ofcollagen threads.
 12. The prosthesis of claim 11 wherein said collagenthreads are arranged to form a felt, a bundle, a wave or a braid. 13.The prosthesis of any claims 10–12 wherein said collagen fibers orthreads are partially or completely crosslinked.
 14. The prosthesis ofclaim 1 wherein said prosthesis additionally contains an anticoagulant,one or more antibiotics, or one or more growth factors.
 15. A method forpreparing a prosthesis having two or more superimposed, bonded layers ofcollagenous tissue, comprising: (a) bonding two or more collagen layerstogether using heat welding by heating said collagenous layers for atime and under conditions sufficient to effect the bonding of thecollagen layers and to form a prosthesis; (b) cooling said heatedprosthesis; and (c) crosslinking said prosthesis with a crosslinkingagent that permits bioremodeling, wherein said collagenous tissue isderived from tunica submucosa of the small intestine separated from theother layers of the small intestine, wherein said thus formed prosthesisis non-antigenic, and wherein said thus formed prosthesis when implantedinto a mammalian patient, undergoes controlled biodegradation occurringwith adequate living cell replacement such that the original implantedprosthesis is remodeled by the patient's living cells; wherein thecollagenous tissue layers are sterilized with peracetic acid beforebonding in step (a) or the prosthesis is sterilized after crosslinkingin step (c).
 16. The method of claim 15 wherein said heat welding isfrom about 50° C. to about 75° C.
 17. The method of claim 15 whereinsaid cooling is accomplished by quenching.
 18. The method of claim 15wherein said heat welding is accomplished for a time from about 7minutes to about 24 hours.
 19. The method of claim 15 wherein saidprosthesis is crosslinked with the crosslinking agent1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.
 20. Themethod of claim 15 wherein said heat welding is from about 60° C. toabout 65° C.
 21. The method of claim 15 wherein said heat welding is atabout 62° C.
 22. The method of claim 15 wherein said heat welding isaccomplished for a time of about 1 hour.
 23. A method of repairing orreplacing a damaged tissue comprising implanting a non-antigenicprosthesis in a patient comprising two or more superimposed, bondedlayers of collagenous tissue derived from tunica submucosa of the smallintestine separated from the other layers of the small intestine whichhave been sterilized with peracetic acid and crosslinked with acrosslinking agent that permits bioremodeling, wherein two or morelayers of the prosthesis are completely bioremodelable, and which, whenimplanted into a mammalian patient, undergoes controlled biodegradationoccurring with adequate living cell replacement such that the originalimplanted prosthesis is remodeled by the patient's living cells.
 24. Amethod for preparing a non-antigenic prosthesis prepared fromcollagenous tissue derived from tunica submucosa of the small intestineseparated from the other layers of the small intestine comprising: (a)sterilizing the collagenous tissue with peracetic acid at aconcentration between about 0.01 and 0.3% in water; and (b) crosslinkingsaid sterilized collagenous tissue with a crosslinking agent thatpermits bioremodeling; wherein a prosthesis is formed from two or moresuperimposed, bonded layers of collagenous tissue, wherein two or morelayers of the prosthesis are bioremodelable, and wherein the prosthesiswhen implanted into a mammalian patient, undergoes controlledbioremodeling occurring with adequate living cell replacement such thatthe original implanted prosthesis is remodeled by the patient's livingcells without eliciting a significant humoral immune response.
 25. Themethod of claim 24 wherein the prosthesis is sterilized with peraceticacid prior to implantation into the mammalian patient.